The long range goal of this project is to provide an approach to therapy for certain hereditary anemias of man including Cooley's anemia and sickle cell anemia. The pathophysiology of these disorders has been elucidated to a significant extent in recent years. The induction of gamma globin synthesis or the introduction of Beta A or gamma globin genes into the cells of patients with these hereditary disorders may lead to successful therapy. Knowledge of the basic control of human globin syntehesis at the gene level is critical for this approach. The isolation of biologically active human globin mRNA and the availability of a complementary DNA which to a great extent faithfully encodes the nucleotide sequence of globin mRNA provides a unique opportunity to pursue the isolation and characterization of human globin genes from native DNA in human cells. Specific alpha, beta and gamma mRNAs will be isolated and cDNAs prepared using these mRNAs as templates. During the past year, the number of alpha, beta and gamma globin genes present in non-thalassemic and thalassemic cell DNA has been quantitated. These studies indicate that there is a great specificity in the association of globin cDNA with specific globin mRNAs and with specific native globin genes present in cellular DNA. Because of this specificity, globin cDNA can be used as a specific assay in the isolation and purification of specific globin genes. The availability of restriction enzymes which cut DNA at specified sites only will provide uniform DNA fragments for further purification and analysis. In addition, the isolation of native globin DNa will take advantage of the specific interaction between globin mRNA and native globin genes. The isolated globin genes will be characterized biologically as well as chemically by their ability to serve as templates for bacterial and mammalian RNA polymerases in in vitro systems. In addition, attempts will be made to introduce globin DNA intro fibroblasts, frog oocytes, and erythroid percursor cells in an attempt to determine the effect of globin genes in these cells and the possible integration of this added DNA. The possibility that globin DNA introduced into cells might derepress globin genes which are present but unexpressed in thalassemia cells will also be tested.